Fig. 4: ETMR cellular heterogeneity was recapitulated in ETMR-forebrain organoids.

A Schematic of ETMR-FBO generation via co-aggregation of hiPSCs with murine or human ETMR cells: i) on day 0, hiPSCs were co-aggregated with ETMR cells; ii) organoids were matured under standard conditions; iii) on day 30, ETMR-harboring (ETMR-FBO) and non-harboring (FBO) organoids were used for histology, immunostaining, and scRNA-seq. Created with BioRender.com. B Representative maximum projection image of all confocal planes from whole-mount immunostained (WMI) FBOs for DAPI and MAP2 (neuronal maker) (n = 6 organoids; 1 experiment). Scale bar = 200 μm. C Representative WMI image of YFP+ murine ETMR cells integrated in FBOs (mETMR-FBO) (n = 36 organoids; 2 experiments). Scale bar = 200 μm. D (a–o) Immunohistochemistry (IHC) of mETMR-FBO, hETMR-FBO, and FBO (1x FFPE block with ~ 20 organoids/condition; 2 experiments); a, f, k) H&E staining of each condition (×4 magnification, scale bar = 250 μm). Squares indicate areas further characterized by IHC; (a–e). Tumoral regions in mETMR-FBO showed high cell density, Nestin positivity, β-Catenin-accumulation (red arrows) and LIN28A negativity¹⁸; (f-j), tumor areas in hETMR-FBO showed Nestin and LIN28A positivity (×20 magnification, scale bar = 50 μm). E–K scRNA-seq analysis of murine and human ETMR-FBO. E Integrated UMAP clustering from one FBO and two mETMR-FBO, showing malignant (murine), non-malignant (human) and non-assigned (NA) cells. F UMAP showing main FBO cell types. Colored by cell types (RG radial glia, NProg neuroprogenitor, Nb neuroblast, Nb_ex Nb_excitatory, Nb_Inh, Nb_inhibitory). G UMAP of mETMR-FBO showing ETMR subpopulations (RG-like, NProg-like, and Nb-like), colored by cell type. H Pie charts of ETMR subpopulation proportions across datasets (primary murine/human ETMR, mETMR-FBO, and 2D ETMR culture). Same color code as in (G). I–K UMAPs from integrated clustering of FBO and hETMR-FBO (n = 1 sample each), showing hETMR and FBO cells (I), FBO cell types (J), and hETMR subpopulations (K) recapitulated within FBOs. Source data of H is provided as a Source Data file. Sample type symbol in D–K created with BioRender.com.